Lipid rafts are membrane microdomains that have been proposed to restrict membrane protein mobility in the plasma membrane, thereby resulting in their local concentration. Our findings that MHC-II associates with lipid rafts in antigen processing compartments and that lipid raft association of MHC-II is important for CD4 T cell activation led us to investigate the importance of membrane lipids in MHC-II transport and peptide binding. We have found that pMHC-II associates with members of the tetraspanin family that reside in tetraspan membrane microdomains and our future work will explore the importance of these interactions for MHC-II transport, membrane microdomain localization, and APC function using mice deficient in tetraspanin proteins. Newly synthesized MHC-II binds to a protein termed Invariant chain (Ii) in the endoplasmic reticulum, and it is widely thought that MHC-II trafficking to antigen processing compartments is directed by endo/lysosomal sorting signals present in Ii. Nevertheless, we and others have shown that Ii-free pMHC-II is capable of endocytosis from the plasma membrane and that these complexes can access antigen processing compartments. We are investigating the machinery that regulates endocytosis, recycling, and endo/lysosomal sorting of both Ii-associated MHC-II and Ii-free pMHC-II in APCs in an attempt to determine the functional consequences of endocytosis of these two distinct types of MHC-II. In APCs, antigen processing compartments often have the phenotypic characteristics of multivesicular bodies (MVB). In studies examining MHC-II trafficking to antigen processing compartments we found that activated (but not resting) B cells secrete significant amounts of their total pool of pMHC-II on MVB-derived vesicles termed exosomes. Surprisingly, it was predominantly Ii-free surface pMHC-II that internalized and trafficked to these MVB, highlighting a previously unrecognized transport pathway followed by pMHC-II. We also found that interaction of antigen-loaded B cells with antigen-specific T cells stimulates exosome release from B cells, and these exosomes in turn can stimulate primed (but not nave) T cells to proliferate. Our results support a model that T cell stimulated exosome release from activated B cells serves to augment T cell responses. The movement of proteins and lipids from one intracellular compartment to another is carried out by a well-orchestrated process of transport vesicle formation, vesicle docking with a target compartment, and finally vesicle-target membrane fusion. The proteins that catalyze membrane fusion are termed SNAREs. During our work investigating MHC-II trafficking we identified novel SNARE proteins that could potentially affect intracellular transport of not only MHC-II but other membrane-associated proteins in immune cells. We have been studying the role of these SNAREs in MHC-II trafficking and in the regulation of protein transport in immune cells with the goal of elucidating the role that distinct SNARE isoforms play in regulating protein traffic in APCs. We have found that SNARE membrane fusion complexes are enriched in lipid raft plasma membrane microdomains that regulate exocytosis in mast cells. We have also been investigating the role of distinct SNARE isoforms in regulating secretory granule exocytosis from mast cells using a variety of SNARE knock-out mice and have found that VAMP-8, but not VAMP-2 or VAMP-3, regulates mast cell secretory granule exocytosis. Surprisingly, VAMP-8-deletion only affects serotonin exocytosis but not histamine or TNF-alpha exocytosis, showing for the first time that secretory granule heterogeneity exists in mast cells.